Formation of Circular Satellite Tobacco Ringspot Virus RNA in Protoplasts Transiently Expressing the Linear RNA

Abstract

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L-(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L-(+)M into the corresponding circular form C-(+)M. We transiently expressed L-(+)M wild type and L-(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C-(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L-(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53-211 and 268-350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L-(+)M. The mutant L-(+)M was found to be extensively ligated to C-(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3' terminal sequence. A trinucleotide in the 3' region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C-(+)M formation. These results suggest that the 3' stem contributes to the suitability of the small L-(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structure in vivo.

Not As They Seem

“…For the encapsidated polarity of sTRSV RNA, the back reaction of autolytic cleavage, i.e., ligation, proceeds to only a very limited extent (36). The in vivo ligation of the encapsidated polarity RNA is apparently enzymic, proceeds without infection by TRSV, and requires only a portion of the sTRSV RNA sequence (13). We demonstrated that the cleavage reactions for both polarities of sTRSV RNA proceed by inversion of configuration about the phosphorus atom (50).…”

cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

“…In potato spindle tuber viroid, which requires host components for cleavage, the switch from cleavage to ligation is driven by a change from a tetraloop to a conformation that resembles the loop E structure found in 5S rRNA (2). Chay et al (8) showed that linear satTRSV RNA folds into a nonhammerhead conformation to facilitate efficient ligation to form a circular molecule. This ligation structure juxtaposes the 5Ј and 3Ј ends rigidly in close proximity via base pairing.…”

“…In most cases monomeric plus strands are ligated into circular forms (45) that are encapsidated in the virions provided by the helper virus. In other examples, the encapsidated form of the satellite RNA is linear (27, 34), and it is circularized only in the cell (8,47,48).Because rolling circle satellite RNAs encode no proteins, the sequence and higher-order structure of the RNA itself must confer the biological properties, as has been shown for other noncoding, infectious RNAs (reviewed in references 19 and 47). The RNA sequence and structures involved in pathogenicity and replication are well characterized for the linear satellite RNAs of Cucumber mosaic virus (19) and Turnip crinkle virus (satRNA C) (42,44,52).…”

“…In most cases monomeric plus strands are ligated into circular forms (45) that are encapsidated in the virions provided by the helper virus. In other examples, the encapsidated form of the satellite RNA is linear (27, 34), and it is circularized only in the cell (8,47,48).…”

“…The RNA sequence and structures involved in pathogenicity and replication are well characterized for the linear satellite RNAs of Cucumber mosaic virus (19) and Turnip crinkle virus (satRNA C) (42,44,52). However, little is known about replication and encapsidation signals on rolling circle satellite RNAs (8).…”